BIOCHEMICAL ANALYSIS OF GENETIC RECOMBINATION IN YEAST

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The long term goal of the proposed research is to understand the molecular and enzymatic mechanisms of genetic recombination. This project will focus on the identification and characterization of the proteins that catalyze homologous recombination in the yeast Saccharomyces cerevisiae. This organism is well suited for these studies since it is easily grown mitotically or meiotically in liquid culture; several mutants are available that have defects in recombination, and, in addition, yeast exhibits high frequencies of homologous recombination. Crude extracts prepared from wildtype and recombination defective strains will be monitored using several different sets of substrates and assays for partial or complete recombination reactions. Mutants strains that show alterations by any of the assays will be used for in vitro complementation of these activities. Activities that catalyze partial recombination reactions will be purified by fractionation of crude lysates. The purified proteins will be used to reconstitute a complete recombination reaction in vitro. An understanding of the mechanisms of homologous recombination may lead to insights into health-related problems. For example, the activation of some cellular onc genes in mammals is frequently associated with translocations that may occur by recombination between repeats on non-homologous chromosomes. Improved knowledge of homologous recombination may also be useful for gene therapy.